PROJECT SUMMARY The cell surface CD47 protein is a marker of ?self? and signals through its receptor, signal-regulatory protein ? (SIRP?) expressed by macrophages, to transmit an anti-phagocytic (?don't-eat-me?) signal. CD47 is expressed by virtually all cell types, is overexpressed by cancer cells as a mechanism to avoid immune surveillance and phagocytosis, and is a negative prognostic factor for survival. Blocking this interaction is of therapeutic interest, because it results in increased cancer cell phagocytosis (innate immunity) as well as priming of a cytotoxic antitumor T-cell response (adaptive immunity). However, due to ubiquitous CD47 expression, anti-CD47 immunotherapies must overcome a large antigen sink (reduced bioavailability), and are limited by on-target/off-tumor toxicity, such as thrombocytopenia and anemia. In contrast, the receptor SIRP? is mainly expressed on myeloid cells, such that inhibitors targeting SIRP? will have reduced toxicity and increased bioavailability. SIRP? is a transmembrane protein composed of a conserved cytoplasmic tail region and three N-terminal extracellular immunoglobulin superfamily (IgSF) domains: one V-set and two C1-set IgSF domains. The related family members SIRP?and SIRP?share homologous IgSF domains, but have distinct cytoplasmic domains and signaling properties. The region of SIRP? that interacts with CD47 is contained within the soluble 118-amino acid N-terminal IgSF V-set domain, making synthesis of this target very feasible. Using a drug discovery platform that employs enantiomeric screening (mirror-image phage display) coupled with protein design, we have successfully developed protease-stable D-peptide entry inhibitors of HIV, RSV, and Ebola viruses. These inhibitors bind pockets that are similarly involved in functionally critical protein- protein interactions, traditionally deemed undruggable by small molecule approaches. Our anti-HIV D-peptide entry inhibitor is in advanced preclinical trials and was trimerized to simultaneously bind all three pockets on HIV's trimeric envelope protein, resulting in extremely high (pM) potency. In this 1-year grant application, we propose to discover D-peptides that specifically bind only to SIRP? and inhibit its interaction with CD47, and then characterize their binding profile on SIRP?and SIRP?. D-peptides typically display extended in vivo half-lives and low immunogenicity, and their small size gives them the potential for enhanced tumor penetration. Also, the chemical synthesis of these D-peptides will result in a much more cost-effective cancer immunotherapeutic compared to biologics. Success here will warrant subsequent structural characterization, affinity maturation, testing in cell culture and animal models, and IND-enabling toxicology studies on our D-peptide inhibitors of SIRP? to aid in the treatment of cancer.